The invention concerns a nuclear magnetic resonance (NMR) method for singlet-state exchange NMR-spectroscopy.
Long-lived singlet-states offer unique opportunities for studying very slow dynamic processes in solution-state NMR. A suite of pulse sequences is presented which can achieve broadband excitation of singlet-states in pairs of coupled spins. The most elaborate of these experiments, two-dimensional singlet-state exchange spectroscopy (SS-EXSY) is independent of the offsets of the two spins, their relative chemical shifts, and their scalar couplings. The new methods afford the study of very slow translational diffusion, chemical exchange or refolding processes, and may be suitable to observe cross-relaxation processes between singlet states using mixing times on a scale τm≈TS>>T1. The lifetimes TS of singlet-states of pairs of protons in a partially deuterated saccharide are shown to be longer by a factor of ˜23 than the longitudinal proton relaxation times T1 in the same compound.
In conventional NMR studies of slow translational diffusion, chemical exchange, and refolding of biomolecules such as proteins or nucleic acids, the upper limit of the accessible time-scale is normally determined by the longitudinal relaxation time constant T1, which is usually regarded as the maximum life-time of the memory of nuclear spins. NMR could not monitor correlations between states that are connected through very slow processes with a characteristic time constant longer than T1 so far. However, recent work by Caravetta and Levitt [1; 2] has shown that it is possible to excite and observe so-called singlet states in systems containing pairs of scalar-coupled spins. Such singlet states| |S0>=N{|αβ>−|βα>} with N=2−1/2 are antisymmetric under a permutation of the two spins, and the singlet-state life-time TS is not affected by the mutual dipole-dipole interaction between these two spins. Singlet-state life-times TS can be more than an order of magnitude longer than longitudinal relaxation times T1 in two-spin systems with analogous dynamic properties [1; 3]. A non-vanishing population of a singlet state |S0> can be obtained by first exciting a zero-quantum coherence ZQx, i.e., a coherent superposition of two states |αβ> and |βα> with a suitable phase, which is then converted into a population of the singlet state |S0> by an appropriate radio-frequency irradiation that suppresses the effects of the offsets [4]. In practice, this irradiation converts a weakly or strongly coupled two-spin IS system (JIS≠0) into an I2 system with two magnetically equivalent spins. Alternatively, as shown below, a singlet state |S0> can be populated by first creating longitudinal two-spin order σ=2IzSz, which, in contrast to a system in thermal equilibrium with σ=Iz+Sz, comprises eigenstates |αβ> and |βα> with non-vanishing populations. Like a ZQx coherence, a 2IzSz, term (also known as ‘ZZ order’), can be converted into a population of the singlet-state |S0> by appropriate radio-frequency irradiation, which in effect leads to decoupling of the JIS interaction. The population of a singlet state can in principle be preserved indefinitely if decoupling is ideal and if all relaxation mechanisms other than the dipolar interaction between spins I and S can be neglected. In practice, non-ideal decoupling leads to a reduction in the life-time TS [5], as does relaxation of the I and/or S spins by chemical shift anisotropy (CSA) or by dipolar interactions with further spins in the vicinity that may belong to the same molecule or to neighboring (solvent) molecules.
It has been shown that singlet states can be exploited to study slow translational diffusion [3]. Preliminary demonstrations have been carried out with a simple test molecule, 2-chloroacrylonitrile, which contains only two protons I and S with a small difference in chemical shifts ΔνIS=νI−νS=38 Hz at 300 MHz (0.13 ppm) and a scalar coupling JIS=−3 Hz [3; 6]. We have found that, contrary to earlier belief; proton-containing solvents do not lead to a dramatic reduction of the lifetime TS of the singlet states. Molecules such as saccharides that contain more protons have reduced life-times TS compared to molecules that contain only isolated proton pairs, but partial deuteration of saccharides in all positions except for the H5′ and H5″ protons leaves a pair of diastereotopic protons with JIS=J(H5′ H5″)=−12.5 Hz and a small difference in chemical shifts ΔνIS=νI−νS=ν(H5′)−ν(H5″)=75 Hz at 400 MHz (0.18 ppm). (Such partial deuteration can be achieved conveniently by oxidation of a perdeuterated saccharide to an aldose and subsequent reduction.) When these saccharides are incorporated into nucleic acids such as RNA, the chemical shifts of the H5′ and H5″ protons should be affected by conformational exchange and refolding processes [7; 8]. Such protons can therefore be used to study the kinetics of slow exchange, for example by two-dimensional exchange spectroscopy (EXSY) [9; 10]. The kinetic window of such experiments is normally limited by longitudinal relaxation to mixing times τm≈T1(1H). In 1H-detected 15N exchange spectroscopy [7] this limitation can be somewhat relaxed since one can use mixing times τm≈T1(15N)>T1(1H). One of the objectives of this paper is to show that one can design singlet-state (SS) variants of two-dimensional exchange spectroscopy (SS-EXSY) that allow one to extend the kinetic window to mixing times τm≈TS(1H)>T1(15N)>T1(1H).
The pulse sequences for singlet-state excitation that have been described so far [3; 4] suffer from a number of drawbacks: (i) the RF carrier νRF must be positioned half-way between the chemical shifts νI and νS of the two nuclei, (ii) the efficiency of the sequences depends on the difference ΔνIS=νI−νS between the chemical shifts, and (iii) the efficiency also depends on the scalar coupling constant JIS. Clearly, slow dynamic processes AB, such as chemical exchange or refolding of biomolecules like proteins and nucleic acids, must lead to changes in chemical shifts νIA≠νIB and/or νSA≠νSB to be observable by NMR. In general, the differences in chemical shifts may also be affected by chemical exchange, i.e., ΔνISA=(νIA−νSA)≠ΔνISB=(νIB−νSB). Furthermore, it is possible that the scalar couplings are also affected by chemical exchange, i.e., JISA≠JISB. If singlet states are to be used to investigate such slow processes, the pulse sequences must be modified so as to become independent of chemical shifts and couplings.